PROJECT SUMMARY Adenoid cystic carcinoma (ACC) is the second most common malignancy of the salivary glands. Over 60% of the ACC patients succumb to the disease within 15 years from diagnosis. The lack of reliable experimental models for ACC has limited our progress in understanding the biology of the disease and the proliferation of preclinical studies to test new therapies. In this proposal we address this fundamental need by proposing the generation of genetically engineered mouse models that develop autochthonous ACCs. These mice will help determine the role of molecular alterations frequently found in human ACCs and will generate an in vivo platform for molecular and preclinical studies that will help advance towards more effective therapies to treat ACC patients. MYB is by far the most commonly altered gene in human ACCs, as over 70% of the tumors overexpress MYB-NFIB fusions or the full-length MYB. Our preliminary studies show that MYB overexpression in transgenic mice induces ACCs with long latency, indicating that MYB promotes ACC development, but also suggesting that additional alterations are required for ACC development. Genetic alterations affecting MYB-unrelated genes are found at lower frequencies in human ACCs. Notably, mutations in different genes that are predicted to result in activation of NOTCH signaling, including activating mutations in NOTCH1, were found in 25%-35% of the human ACCs, associated with poor prognosis. Of those, inactivating mutations in the SPEN gene were found in ~20% of the ACCs. SPEN is a transcriptional repressor of NOTCH signaling and functions as a tumor suppressor through NOTCH-dependent and NOTCH-independent mechanisms. Importantly, these mutations co-exist with MYB alterations, but their contribution to ACC development is presently unknown. In this proposal we will analyze the cooperation of MYB with NOTCH activation or SPEN inactivation during ACC development, in mouse models that allow the activation of mutations in salivary glands. The inducible nature of the system used to activate these alterations will allow us to determine whether they are required to maintain tumor growth and to identify mechanisms involved in tumor regression. Analysis of the transcriptomes of the tumors that develop in these mice will allow us to identify MYB-regulated genes and pathways that contribute to ACC development, some of which may be required to maintain tumor growth, and thus could be excellent targets to explore new therapies for ACC.